Project Summary/Abstract The genome replication is fundamental process of life that impacts virtually every aspect of human health. Therefore, a detailed understanding of genome replication mechanisms is vital for future advances in disease diagnosis, drug design, and patient treatment. The bulk of human DNA replication is performed by the B-family DNA polymerases Pol? and Pol?. However, Pol? and Pol? cannot begin synthesis without DNA primers. To circumvent this problem, a specialized RNA polymerase called primase generates the initial primers. Then, a dedicated B-family DNA polymerase ? (Pol?) working in a tight complex with primase (referred to as a primosome) extends the RNA primers with deoxyribonucleotides, before switching them to Pol? for the start of leading-strand replication and to Pol? for the start of replication of each of the millions of Okazaki fragments of the lagging strand. The remaining member of the B-family is DNA polymerase ? (Pol?), which is a key player in translesion DNA synthesis. A significant gap remains in the current understanding of B-family DNA polymerases' function, especially regarding the key factors that tightly coordinate polymerase transactions at the replication fork. The crucial components of this global coordination are the mechanisms of template:primer handover from Pol? to Pol? and Pol? during asymmetrical synthesis of both the leading and lagging strands, counting the length of Okazaki fragments, and the switch of Pol? and Pol? to productive elongation. We discovered that the accessory B-subunit of Pol? also makes a complex with the catalytic subunit of Pol?, which is important for the polymerase switch during lesion bypass. However, the mechanism of this switch remains unknown. One of the biggest impediments in resolving these challenges is insufficient structural information, especially for entire polymerase complexes, including Pol?, Pol?, and Pol?, as adequate knowledge of molecular structure is essential for the design of meaningful functional assays. Based on our previous productive studies of primosome and the components of Pol?, Pol?, and Pol?, we propose a new direction of investigation that examines the tightly coordinated events in primer synthesis, primer handoff from Pol? to Pol? and Pol?, and their switch to accurate elongation mode. For the proposed studies, we will apply X-ray crystallography and a variety of structure-guided biochemical and single-molecule experiments. Most of these studies will be conducted using the in vitro reconstituted human replisome.